The objective of this proposal is to study the replication of a eukaryotic gene with a known transcriptive function. The genes which code for 18S and 28S ribosomal RNA in the frogs, Xenopus laevis and Xenopus borealis were selected as the system of choice for three reasons: 1) They are readily isolatable in neutral CsCl gradients. 2) They occur as tandemly repeated sequences. 3) Their structure and organization have already been extensively studied. Several specific questions will be asked relating to the replication of these genes in each of the organisms: 1) Does DNA synthesis begin at a unique site within a repeating unit? 2) If so, where within the repeat is the replication initiation site located? 3) Do adjacent repeating units begin replication synchronously, or can one initiate DNA synthesis while its neighbor remains dormant? 4) If defined replication origins exist, what is the nucleotide sequences around the origin? 5) How similar or different are these sequences in Xenopus laevis and Xenopus borealis? The source of DNA for these experiments will be stage 7 Xenopus blastulae. At this stage the S pahse lasts about 10 minutes so that DNA isolated from these embryos should be enriched in replicating molecules. A second source of DNA will be from synchronized cultured Xenopus cells at the time in the S phase when the ribosomal DNA is replicating. The isolation of ribosomal DNA will be accomplished by isopycnic centrifugation in neutral CsCl and analysis of replicating ribosomal DNA molecules will be performed by electron microscopy before and after cleavage with restriction endonucleases.